Field of Invention
The invention pertains to the agricultural sector, specifically to biopesticides for biological control of pests.
Background of the Invention
Baculoviruses (from Latin baculum=staff) are viruses that specifically infect insects, mainly members of orders Lepidoptera, Hymenoptera and Diptera (Wang & Jehle, 2009; Duffy et al., 2006; Herniou et al., 2003; Boucias et al., 1998). They are found in the environment as occlusion bodies (OBs) (Slack & Arif, 2007), which are protein structures that protect the virions and grant them increased endurance in the environment.
The Baculoviridae family is characterized by the fact that its members contain a circular, double-stranded DNA genome, ranging from 80 to 180 kilobase pairs (kbp). The family comprises four genera, classified according to their structural, molecular and biological characteristics: alphabaculovirus consists of nucleopolyhedroviruses (NPVs) isolated from lepidopterans, betabaculovirus consists of granuloviruses (GVs) isolated from lepidopterans, deltabaculovirus consists of NPVs isolated from dipterans, and gammabaculovirus consists of NPVs isolated from hymenopterans (ICTV, 2012; Jehle et al., 2006; Miele et al., 2011).
These genera are distinguished by their structural characteristics, such as the morphology of their occlusion bodies as well as by some biological aspects (Herniou et al., 2004; Jehle et al., 2006). Throughout the infection cycle, the formation of two viral phenotypes has been observed: budded virions (BVs) and occlusion-derived virions (ODVs) (Theilmann et al., 2005). The two phenotypes have similar nucleocapsid structures and contain identical genetic information, but the source and composition of their envelopes is different (Funk et al., 1997; Braunagel & Summers, 1994). BVs are responsible for effecting the systemic infection of the host, while ODVs are responsible for spreading the virus between hosts. Baculoviruses are found in the environment as occlusion bodies (OBs); said bodies keep them viable for prolonged periods of time. While the OBs of GVs normally contain only one (1) virion, the OBs of NPVs may contain multiple virions, and furthermore, said virions may in turn contain multiple nucleocapsids. (FIG. 1). There are various studies concerning the use of Baculoviruses as regulation factors of some populations of insects, especially those that affect agricultural production. This technology offers considerable advantages over chemical pesticides, including the reduction of toxic residues in food products and soil, as well as lower health hazards.
WO2014/182228 describes a method for increasing the insecticidal efficacy of a pathogenic microorganism (Spodoptera frugiperda nucleopolyhedrovirus (SfNPV)) by means of its association with a mutualistic organism (yeast species of the Metschnikowia genus, such as Ascomycota and Sacharomycetes; yeast species of Candida; species of Cryptococcus and species of Pseudozyma) that co-exists in intimate association with the larva and that stimulates the ingestion of the pathogen by said larva. However, the drawback of said method is that during the storage period, the metabolic activity of the mutualistic organism can continue, thereby producing metabolites that alter viral activity, and thus, the quality of the product.
WO2011/117411 titled “Entomopathogenic Viral Preparation”, discloses a composition containing at least one Cydia pomonella granulovirus (characterized by a specific restriction fragment pattern), used to control the codling moth larva Cydia pomonella (Lepidoptera:Tortricidae) and the Oriental peach moth C. molesta. The composition disclosed therein does not have any application for the control of migratory pests from tropical and subtropical zones of America, such as the fall armyworm S. frugiperda. 
In addition to the formulations and combinations of aforementioned agents, some studies have focused on the genetic diversity of the control agents against specific pests, with the goal of selecting highly pathogenic isolates. Patent WO2014/161974 relates to new genotypes of Chrysodeixis chalcites nucleopolyhedrovirus (Chch SNPV), the process for production thereof and uses thereof as biological control agents. Patent application ES2301352 discloses six new genotypes of S. exigua multiple nucleopolyhedrovirus termed AlPstM0935, AlPstM1400, AlPstM1033, AlPstM1449, AlPstM0923, and AlPstM0657, and uses thereof, preferably as a combination of two or more genotypes, in insecticidal compositions against S. exigua. 
In the last few decades, various strategies to genetically modify Baculovirus have been employed in order to increase its virulence, pathogenicity and/or insecticidal activity, which include the following:                a) insertion of the gene coding for the toxin produced by the scorpion Buthus eupus;         b) insertions of the gene coding for the esterase of the juvenile hormone produced by Heliothis virescens;         c) insertion of the gene for TxP-1 toxin;        d) insertion of the gene for AaIT toxin from the scorpion Androctonus australis into AcNPV (Autographa californica nucleopolyhedrovirus);        e) insertion of the gene for the cry toxin produced by Bacillus thuringiensis;         f) insertion of the gene for diuretic hormone; g) insertion of the scorpion neuroselective neurotoxin LqhIT2;        g) insertion of sequences that express substances that suppress the appetite of Helicoverpa zea and related pests (see Brazilian patent PI0901235-4 and patent U.S. Pat. No. 6,096,304); and        h) modification of the gene for chitinase, to attenuate the lysis of the larva and increase the yield and recovery of virions for mass production.        
Although the genetic modification strategies have been shown to improve the characteristics of the modified baculoviruses, the environmental policies of several European and Latin American countries, including Colombia, do not allow the use of genetically modified organisms, because of which these microorganisms cannot be easily applied on an industrial scale or in crop fields.
For that reason, it has been necessary to develop various types of biopesticidal formulations based on unmodified organisms that are highly specific against the pests faced and that overcome the drawbacks associated with low efficacy and high sensitivity to UV radiation. Thus, in the study titled “Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population”, Corpoica describes the purification of genotypes from a native isolate of S. frugiperda multiple nucleopolyhedrovirus (SfMNPV). This study reports the production of 83 clones, 10 of which corresponded to different genotypes and were subjected to molecular and biological characterization. The results showed that one genotype was more pathogenic.
The study also demonstrated that the combination of this more pathogenic genotype (termed A) with three other selected genotypes (termed C, D, and E) exhibited a lower or equal potency to that of the wild-type virus. The conclusion of said study was that the co-occluded combination of various genotypes in the wild is a pathogenicity regulation mechanism to ensure the persistence of the pathogen in nature and may result in lower insecticidal activity than the original wild-type virus (Barrera et al., 2013).
Additionally, in another study titled “Eudragit S100® microparticles containing Spodoptera frugiperda nucleopolyhedrovirus: Physicochemical characterization, photostability and in vitro virus release”, Corpoica reports a method for microencapsulation of occlusion bodies of the NPV001 isolate from S. frugiperda nucleopolyhedrovirus, using a Eudragit S100® methacrylic acid polymer, by means of the emulsion and solvent evaporation method. The microcapsules produced exhibited a loss of efficacy after 2 hours of irradiation with ultraviolet light (Villamizar et al., 2010), possibly due to the absence of other formulation aids and to the fact that the active ingredient is a complete wild-type virus.
In a later study titled “Microencapsulation of a Colombian Spodoptera frugiperda Nucleopolyhedrovirus with Eudragit S100® by spray drying”, Corpoica reports a formulation that is similar to the previous formulation, but with an improved microencapsulation process, abandoning the solvent evaporation method in favor of a fluidized bed granulation method (Camacho et al., 2015). In this study, the size of the microcapsules was decreased to 20 μm and photostability to UV exposure increased.
The interaction of the various genotypes that comprise a nucleopolyhedrovirus isolate may have negative, neutral or positive effects. For example, pure genotypes of S. exigua nucleopolyhedrovirus (SeMNPV) exhibited greater insecticidal activity that the complete wild-type virus (Muñoz et al., 1998). However, in a S. frugiperda nucleopolyhedrovirus isolate from Nicaragua, the complete wild-type virus exhibited a greater pathogenicity than the pure genotypes (Simón et al., 2004; 2005). Furthermore, the combination of some genotypes restored the pathogenicity of the pure genotypes, but never achieved a greater pathogenicity than that exhibited by the wild-type isolate.
The inventors designed combinations of two or more pure genotypes cloned from the Spodoptera frugiperda nucleopolyhedrovirus isolate (NPV015 through NPV019) that exhibited a greater pathogenicity than the wild-type genotype.